Specific Aim 1. Mechanisms regulating neurotrophin retrograde signaling and neuronal growth and survival. The neurotrophic signaling pathway from axonal terminals to cell bodies is crucial for dendrite growth and neuron survival. BDNF is one of the well-studied neurotrophic factors regulating dendrite outgrowth and branching. By binding to its receptor TrkB, BDNF triggers the internalization of ligand-receptor complexes into signaling endosomes, and activates signal transduction cascades, ultimately leading to retrograde signaling in the nucleus. While signaling endosome hypothesis is one of accepted models, the molecular machinery that drives retrograde axonal transport of BDNF-TrkB signaling endosomes is largely unknown. It also remains unclear whether retrograde axonal transport of BDNF-TrkB signaling endosomes has a direct impact on dendritic growth in CNS. Dynein motors are responsible for retrograde transport. However, mechanisms recruiting dynein to TrkB signaling endosomes have not been elucidated. In particular, a long-standing question is how BDNF-TrkB signaling complexes are delivered from axonal terminals to cell bodies. Using snapin deficient mice and gene rescue experiments combined with compartmentalized cultures of live cortical neurons, we recently revealed that Snapin, as a dynein adaptor, mediates retrograde axonal transport of TrkB signaling endosomes. Such a role is essential for dendritic growth of cortical neurons. Deleting snapin or disrupting Snapin-dynein interaction abolishes TrkB retrograde transport, impairs BDNF-induced retrograde signaling from axonal terminals to the nucleus, and decreases dendritic growth. Such defects were rescued by reintroducing snapin gene. Our study indicates that Snapin-dynein coupling is one of the primary mechanisms driving BDNF-TrkB retrograde transport, thus providing new mechanistic insights into the regulation of neuronal growth and survival (Zhou et al., Cell Reports, 2012). Specific Aim 2. Retrograde transport regulates autophagy-lysosomal function. Maintaining cellular homeostasis in neurons depends on efficient intracellular transport. Late endocytic trafficking, which delivers target materials into lysosomes, is critical for maintaining efficient degradation capacities via autophagy-lysosomal pathways. However, the mechanisms regulating the autophagy-lysosomal system in neurons remain incompletely understood. Dynein-mediated retrograde transport can enhance late endocytic trafficking to the soma, where lysosomes are predominantly localized, and drive late endosomes and lysosomes close enough to fuse with higher efficiency, thus ensuring proper autophagy-lysosomal function. Our recent study uncovered a critical role for Snapin in regulating late endocytic transport and membrane trafficking (Cai et al., Neuron 2010). Snapin acts as a motor adaptor by attaching dynein to late endosomes. Snapin (-/-) neurons exhibit aberrant accumulation of immature lysosomes, impaired retrograde transport of late endosomes along processes, reduced lysosomal proteolysis, and impaired clearance of autolysosomes, combined with reduced neuron viability and neurodegeneration (Cai and Sheng, Autophagy 2011; Zhou et al., 2011). Specific Aim 3. Anterograde axonal transport regulates synaptic formation and plasticity. The formation of new synapses and remodeling of existing synapses play an important role in the various forms of synaptic plasticity and require the targeted delivery of newly synthesized synaptic components from the soma to the synaptic terminals. Thus, efficient axonal transport of newly synthesized synaptic components to nascent presynaptic boutons is critical in response to neuronal activity. However, the molecular identities of the motor-adaptor complex essential for assembling presynaptic terminals in developing neurons and in remodeling synapses of mature neurons in response to neuronal activity remain unknown. Our previous studies established that syntabulin is an adaptor capable of linking KIF5 motor and synaptic protein cargoes (Su et al., Nature Cell Biology, 2004). Syntabulin-KIF5 mediates axonal transport of synaptic components essential for presynaptic assembly. Syntabulin loss-of-function blocks formation of new presynaptic boutons in developing neurons. Our studies establish that kinesin-mediated anterograde axonal transport is another critical factor in the cellular mechanism underlying activity-dependent presynaptic plasticity (Cai et al., J Neuroscience 2007). Our recent study further demonstrated the critical role of syntabulin in the maintenance of presynaptic function and regulation of synaptic plasticity in well-matured sympathetic SCG neurons (Ma et al., J Neuroscience 2009). Conditional syntabulin knockout mice have been recently generated in the lab. We will use this mouse line to (1) determine whether deficiency in syntabulin/KIF5-mediated transport has any impact on synapse maintenance and plasticity in mature neurons and adult mice; (2) determine whether the motor-adaptor complex regulates the transport in response to synaptic activity; (3) identify the sorting signals for the axon-targeted delivery of presynaptic cargo. In summay, our ongoing study provides new mechanistic insights into (1) how Snapin regulates retrograde axonal transport of neurotrophin signaling endosomes and late endosomal-lysosomal organelles; (2) how syntabulin mediates anterograde transport of presynaptic proteins for synaptic maintenance and plasticity. Our snapin and syntabulin mouse models provide us with unique genetic tools for characterizing the roles of both anterograde and retrograde axonal transport in neurodevelopment and neurodegeneration. These studies will provide genetic evidence as to whether manipulating axonal transport will reduce axonal degeneration, thereby ultimately leading to new therapeutic approaches. Pursuing these investigations will advance our knowledge of fundamental processes that may affect human neurological disorders and is thus the very essence of the mission of the National Institute of Neurological Disorders and Stroke. Related publications from the lab: Qingning Su*, Qian Cai*(equal contributions), Claudia Gerwin, Carolyn L. Smith, Zu-Hang Sheng (2004) Syntabulin: a microtubule-associated protein implicated in syntaxin transport in neurons, Nature Cell Biology 6, 941-953. Qian Cai, Pingyue Pan, and Zu-Hang Sheng. (2007). Syntabulin-kinesin-1 family 5B-mediated axonal transport contributes to activity-dependent presynaptic assembly. Journal of Neuroscience 27, 7284-7296. Huan Ma, Qian Cai, Wenbo Lu, Zu-Hang Sheng (co-corresponding author), and Sumiko Mochida. (2009). KIF5 motor adaptor syntabulin maintains synaptic transmission in sympathetic neurons. Journal of Neuroscience 29, 13019-13029. Qian Cai, Li Lu, Jin-Hua Tian, Yi-Bing Zhu, Haifa Qiao, Zu-Hang Sheng. (2010). Snapin-regulated late endosomal transport is critical for efficient autophagy-lysosomal function in neurons. Neuron 68, 73-86. Qian Cai and Zu-Hang Sheng. (2011). Uncovering the role of Snapin in regulating autophagy-lysosomal Function. Autophage 7, 445-447. Bing Zhou, Yi-Bing Zhu, Lin Lin, Qian Cai, and Zu-Hang Sheng. (2011). Snapin deficiency is associated with developmental defects of the central nervous system Bioscience Report 31, 151-158. Bing Zhou, Qian Cai, Yuxiang Xie, and Zu-Hang Sheng (2012). Snapin recruits dynein to BDNF-TrkB signaling endosomes for retrograde axonal transport and is essential for dendrite growth of cortical neurons, Cell Reports 2, 42-51.